Process for preparing long-chain dicarboxylic acids

ABSTRACT

The present invention particularly discovered strains that are capable of producing a long-chain dicarboxylic acid by culturing microorganisms belonging to  Candida vini Candida entamophila, Candida blankii  and  Pichia farinosa  which has the ability to produce a long-chain dicarboxylic acid in a liquid medium containing a straight-chain saturated hydrocarbon (tridecane) as substrate.

The present invention relates to an advantageous process for producinglong-chain dicarboxylic acids from straight-chain hydrocarbons byutilizing the microorganisms.

BACKGROUND TO THE INVENTION

Several strains of yeast are known to excrete alpha, omega-dicarboxylicacids as a byproduct when cultured on alkanes or fatty acids as thecarbon source. In particular, yeast belonging to the genus Candida, suchas C. albicans, C. cloacae, C. guillermondii, C. interinedia, C.lipolytica, C. maltosa, C. parapsilosis and C. zeylenoides are known toproduce such dicarboxylic acids (Isamu Shiio and RyousukeUchio, Agr.Biol. Chem. 1971, 35: 2033-2042). In C. tropicalis, the first step inthe omega-oxidation pathway is catalyzed by a membrane-bound enzymecomplex (omega-hydroxylase complex) comprised of a cytochrome P450monooxygenase and an NADPH-cytochrome reductase. This hydroxylasecomplex is responsible for the primary oxidation of the terminal methylgroup in alkanes and fatty acids (Michele Gilewicz, Marcelle Zacek,Jean-Claude Bertrand, and Edgard Azoulay, Can. J Microbiol., 1979, 25,201).

It has been established that hydrocarbon substrates are enzymaticallyoxidized in the yeast microsomes. Following transport into the cell,n-alkane substrates for example, are hydroxylated to fatty alcohols by aspecific cytochrome P450 system. Two further oxidation steps, catalyzedby alcohol oxidase and aldehyde dehydrogenase, lead to the correspondingfatty acid. The fatty acids can be further oxidized through the samepathway to the corresponding dicarboxylic acid (Colin Ratledge, J. Am.Oil Chem. Soc., 1984, 61(2), 447-453). The di-terminal oxidation, leadsto dicarboxylic acid production, from aliphatic hydrocarbons by yeasts(Shigeo Ogino, Keiji Yano, Gakuzo Tamura and Kei Arima, Agri. Biol.Chem., 1965, 29(11), 1009-1015).

The omega-oxidation of fatty acids proceeds via the omega-hydroxy-fattyacid and its aldehyde derivative, to the corresponding dicarboxylic acidwithout the requirement for CoA activation. However, both fatty acidsand dicarboxylic acids can be degraded, after activation to thecorresponding acyl-CoA ester, through the β-oxidation pathway in theperoxisomes (Atsuo Tanaka and saburo Fukui, In: The Yeasts, Vol. 3,Metabolism and physiology of Yeasts, Edited by A. H. Rose and J. S.Harrison, 2^(nd) Edition, Academic Press, Harcourt Brace Jovanovich,Publishers), leading to chain shortening. In yeast, beta-oxidation takesplace solely in the peroxisomes (Mitsuyoshi Ueda, Kazunori Yamanoi,Tadashi Morikawa, Hirofumi Okada and Atsuo Tanaka, Agr. Biol. Chem.,1985, 49, 1821-1828).

Dicarboxylic acids produced through fermentation by most yeasts,including C. tropicalis, are often shorter than the original substrateby one or more pairs of carbon atoms and mixtures are common (ShigeoOgino, Keiji Yano, Gakuzo Tamura and Kei Arima, Agr. Biol. Chem., 1965,29(11), 1009-1015., Shio and Uchio, Agr. Biol. Chem., 1971, 35(13),2033-2042). These undesirable by-products are often associated withbiological production of dicarboxylic acids.

It is known that the formation of dioic acids can be substantiallyincreased by the use of suitable mutants (RyousukeUchio and Isamu Shiio,Agri. Biol. Chem., 1972, 36(3), 426-433). The wild-type yeasts producelittle if any dicarboxylic acid. Often, mutants partially defective intheir ability to grow on alkane, fatty acid or dicarboxylic acidsubstrates demonstrate enhanced dicarboxylic acid yields. However, thesemutants have not been characterized beyond their reduced ability toutilize these compounds as a carbon source for growth. In alllikelihood, their ability to produce dicarboxylic acids is enhanced by apartial blockage of the beta-oxidation pathway. Furthermore, compoundsknown to inhibit beta-oxidation (i.e. acrylate) also result in increaseddicarboxylic acid yields.

In addition, the use of such a mutant should prevent the undesirablechain modifications associated with passage through beta-oxidation, suchas unsaturation, hydroxylation, or chain shortening.

Many organisms carry out the transformations, including Cryptococcusneoformans and Pseudomonas aeruginosa, Corynebacterium sp., and at leasttwo strains of Candida, that is C. cloacae and C. tropicalis. (KennethD. Green, Michael K. Turner, Johm M. Woodley, Enzyme and MicrobialTechnology, 2000, 27, 205-211).

The work with the bacterial cells has used wild-type organisms, in whichsolvents, detergents, and immobilization may all give improvedconversions (E. C. Chan and J. Kuo, Biotransformation of dicarboxylicacid by immobilized Cryptococcus cells. Enzyme Microb Technol, 1997, 20,585-589). In contrast, the work with the yeast strains has usually usedmutants of Candida tropicalis in which the β-oxidation of fatty acids isimpaired. Engineered strains of C. tropicalis that lack several keyenzymes of β-oxidation are particularly effective catalysts for theseoxidations. This directs the metabolic flux toward ω-oxidation, and then-alkanes are more efficiently converted to the corresponding dioicacids.

OBJECTS OF THE INVENTION

The main object of the invention is to provide a process for thepreparation of saturated dicarboxylic acids with improved yields andselectivity from the corresponding long chain hydrocarbon usingmicroorganisms.

Other objects of this invention will become apparent from the followingdescription.

SUMMARY OF THE INVENTION

The present invention provides a process for the preparation of one ormore saturated dicarboxylic acids from one or more correspondingsaturated hydrocarbons with the same number of carbon atoms and whereinthe stereostructure of the starting compounds is maintained, comprisingculturing a strain selected from the group consisting of Candida vini,Candida entamophila, Candida blankii and Pichia farinosa in a liquidmedium containing the saturated hydrocarbon as substrate.

In one embodiment of the invention, the concentration of the saturatedhydrocarbon substrate is between 5-15% (v/v).

In another embodiment of the invention, the liquid medium contains metalsalts and organic cofactors.

In another embodiment of the invention, the culturing comprisesbiochemical oxidation carried out at a temperature between 20 and 35° C.and for a period of up to 3 days.

In another embodiment of the invention, the saturated hydrocarbon is astraight chain hydrocarbon with 13 carbon atoms and with a methyl groupat both the terminals.

In another embodiment of the invention, the saturated dicarboxylic acidis recovered from the culture medium by any conventional means.

In another embodiment of the invention, the saturated hydrocarbon istridecane and the corresponding saturated dicarboxylic acid produced isBrassylic acid.

In another embodiment of the invention, the process for producing along-chain dicarboxylic acid comprises culturing a yeast strain selectedfrom the group consisting of Candida vini, Candida entamophila, Candidablankii and Pichia farinosa, capable of assimilating straight-chainhydrocarbons in a liquid medium containing a straight-chain hydrocarbonas a substrate to obtain a fermentation broth containing a long-chaindicarboxylic acid corresponding to said hydrocarbon, and recovering saidsaturated long chain dicarboxylic acid.

In another embodiment of the invention, the culturing is effected underaerobic conditions.

In another embodiment of the invention, the microorganism strain isinitially cultivated in a medium containing a different carbon sourceselected from n-decane and n-dodecane and assimilatable by themicroorganism, following which the saturated hydrocarbon is added to themedium when the microorganism has grown sufficiently, and oxidation iscontinued, under aerobic conditions, to produce the saturateddicarboxylic acid.

In another embodiment of the invention, the pH value of the medium is inthe range of 6.0 to 7.0 in the initial stage of culturing and then at7.0 in the succeeding state of culturing.

In another embodiment of the invention, one or more yield inducersselected from the group consisting of H₂O₂, alkanes, and mixturesthereof are added to the liquid medium.

In another embodiment of the invention, the liquid culture medium isextracted with hexane to remove unwanted matter therein such asunreacted hydrocarbon, by-products such as monocarboxylic acid, areeffectively removed leaving the said dicarboxylic acid in fermentationbroth, followed by addition of an alkaline solution such selected fromthe group consisting of sodium hydroxide or potassium hydroxide to thefermentation broth to adjust pH of the solution to 10 to 13, preferably11 to 12, to dissolve the dicarboxylic acid in said fermentation broth,followed by adjusting the pH of the fermentation broth to pH 2., using amineral acid selected from the group consisting of hydrochloric acid,sulfuric acid, phosphoric acid, and bromic acid, to precipitate thedissolved dicarboxylic acid, followed by extraction of the dicarboxylicacid with ether.

In another embodiment of the invention, the culture medium for growth ofthe microorganism comprises 3.0 g yeast extract, 3.0 g malt extract, 5.0g Peptone, 10 g Glucose and 1 L Distilled water, pH 7.0.

In another embodiment of the invention, the culture medium for testingfor medium production of Dicarboxylic acid comprises 10 g (NH₄)₂HPO₄, 2g K₂HPO₄, 0.3 g MgSO₄, 10 mg FeSO₄.7H₂O, 8 mg ZnSO₄.7H₂0, 8 mg MnSO₄, 9ml Tridecane and 900 ml Distilled water, pH 7.0.

In another embodiment of the invention, the culture medium forpreparation of resting cells and seed culture for fermentation comprises20 g D-Sorbitol, 10 g (NH₄)₂HPO₄, 2 g K₂HPO₄, 0.3 g MgSO₄, 10 mgFeSO₄.7H2O, 8 mg ZnSO₄.7H₂0, 8 mg MnSO₄, 2 g Yeast extract, 100 μg ofBiotin and 900 ml Distilled water, pH 7.0.

In another embodiment of the invention, the culture medium forfermentation comprises Tridecane 5% v/v, Sodium acetate 5 g, K₂HPO₄ 2 g,MgSO₄ 0.3 g, FeSO₄.7H₂O 10 mg, ZnSO₄.7H₂0 8 mg, MnSO₄ 8 mg, Yeastextract 2 g and Distilled water 900 ml, pH 7.0.

In another embodiment of the invention, the selectivity with Pichiafarinose for brassylic acid by oxidation of tridecane is 73.81% and thepercentage of conversion is 21.59.

In another embodiment of the invention, the selectivity with Candidablankii, for brassylic acid by oxidation of tridecane is 45.68% and thepercentage of conversion is 5.22.

In another embodiment of the invention, the selectivity with Candidavini, for brassylic acid by oxidation of tridecane is 99.18% and thepercentage of conversion is 2.80.

In another embodiment of the invention, the selectivity with Candidaentamophila, for brassylic acid by oxidation of tridecane is 23.58% andthe percentage of conversion is 28.70.

In another embodiment of the invention, the culture medium includes oneor more additional nutrients selected from a nitrogen source and aninorganic salt.

In another embodiment of the invention, the nitrogen source is selectedfrom the group consisting of peptone, urea, ammonium phosphate, ammoniumchloride, ammonium sulfate and ammonium nitrate.

In another embodiment of the invention, the inorganic salt is selectedfrom the group consisting of phosphates, sulfates and hydrochlorides ofsodium, potassium, magnesium, iron, nickel and zinc, selected in turnrespectively from the group consisting of KH₂PO₄, K₂HPO₄, Na₂HPO₄.12H₂O, MgSO₄7H₂ O, FeSO₄.7H₂ O, ZnSO₄.7H₂O and NaCl.

In another embodiment of the invention, one or more conventionalnutrients selected from the group consisting of yeast extract, meatextract and D-biotin are added to the culture medium for assisting thegrowth of the yeast.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed in this invention is a process for producing a long-chaindicarboxylic acid by culturing microorganisms belonging to Candida viniCandida entamophila, Candida blankii and Pichia farinosa which have theability to produce a long-chain dicarboxylic acid in a liquid mediumcontaining a straight-chain saturated hydrocarbon (tridecane) assubstrate.

This invention relates to a process for the preparation of a saidsaturated dicarboxylic acid from a saturated hydrocarbon, by the use ofsaid yeast strains.

In an embodiment of the present invention, for producing a long-chaindicarboxylic acid which comprises culturing a yeast belonging to Candidavini Candida entamophila, Candida blankii and Pichia farinosa, capableof assimilating straight-chain hydrocarbons in a liquid mediumcontaining a straight-chain hydrocarbon as substrate to obtain afermentation broth containing a long-chain dicarboxylic acidcorresponding to said hydrocarbon.

The carbon source that is employed as the starting material for thepreparation of saturated dicarboxylic acid is a saturated hydrocarbonhaving 13 carbon atoms.

According to the invention, the saturated hydrocarbon is employed as thecarbon source for the cultivation of the aforementioned microorganism,under aerobic conditions, to produce a saturated dicarboxylic acidhaving 13 carbon atoms. Alternatively, the microorganism can beinitially cultivated in a medium containing a different carbon sourceassimilable by the microorganism, rather than the above-mentionedsaturated hydrocarbon, such as n-decane, n-dodecane. The said saturatedhydrocarbon is then added to the medium when the microorganism has grownsufficiently, and cultivation is continued, under aerobic conditions, toproduce the saturated dicarboxylic acid.

In yet another embodiment of the present invention, resting cells of theaforementioned yeasts can be employed for the preparation of thesaturated dicarboxylic acid in the following manner. First, themicroorganism is cultivated in a medium containing a carbon sourceassimilable by the microorganism and different than the saturatedhydrocarbon, i.e., the starting compound. Subsequently, themicroorganism is cultivated in a medium containing a saturatedhydrocarbon i.e., medium B which is the testing medium for theproduction of dicarboxylic acid, thus-cultivated microorganism istransferred to medium D, for preparation of resting cells and seedculture for fermentation experiments. The resting cells are employed forthe enzymatic oxidation of the said saturated hydrocarbon to obtain thesaturated dicarboxylic acid. This reaction can be carried out bysuspending the microorganism, after it has been removed from the mediumD and addition of an appropriate amount of the saturated hydrocarbon formedium E, the fermentation medium. In the process where the restingcells of the microorganism are used, the cultivation of themicroorganism and the oxidation of the said saturated hydrocarbon can beconducted separately. In the oxidation step, the said microorganism canno longer be cultivated because of no carbon source. The said startingmaterial used in the oxidation step, the saturated hydrocarbon, is notused for cultivation of the microorganism.

The said saturated hydrocarbon is preferably maintained in contact withthe aqueous phase and the microorganism by vigorous stirring or shakingwhich is generally satisfactory, because the said saturated hydrocarbonis a liquid. If needed, sufficient contact can be accomplished byaddition of a surface active agent or the like.

The cultivation process of the invention is carried out in a mediumcontaining the aforementioned carbon source and other conventionalnutrients, such as a nitrogen source and an inorganic salt. Examples ofsuitable nitrogen sources include organic and inorganicnitrogen-containing compounds, such as peptone, urea, ammoniumphosphate, ammonium chloride, ammonium sulfate and ammonium nitrate.Examples of the inorganic salts include phosphates, sulfates andhydrochlorides of sodium, potassium, magnesium, iron, nickel and zinc,such as KH₂PO₄, K₂HPO₄, Na₂HPO₄.12H₂O, MgSO₄7H₂O, FeSO₄.7H₂O, ZnSO₄.7H₂Oand NaCl. Moreover, other nutrients, such as yeast extract, meat extractand D-biotin, can be added to the medium for assisting the growth of theyeast.

The cultivation is carried out at room temperature or at a temperatureslightly higher than room temperature. A temperature in the range of 20°C. to 35° C. is preferred. The pH of the medium is within the range of6.0 to 7.0 in the initial stage of culturing and then at 7.0 in thesucceeding state of culturing, the pH is adjusted by addition of aneutralizing agent, such as ammonia, sodium hydroxide or potassiumhydroxide.

The cultivation is, moreover, carried out under aerobic conditions suchas with shaking or stirring under aeration. These procedures can bringabout satisfactory contact between the saturated hydrocarbon used as thestarting compound, the liquid culture medium and the air phase.

In the cultivation as described above, the said microorganism is grownin order to oxidize the said saturated hydrocarbon so that the saidsaturated dicarboxylic acid accumulates in the culture medium.

When D-biotin is introduced as a nutrient, and the said saturatedhydrocarbon, together with another carbon source, such as D-sorbitol,the microorganism grows initially on the carbon source rather than onthe saturated hydrocarbon. The resting cells medium is with D-biotin,and D-sorbitol, thus-grown microorganism then begins oxidation of thesaturated hydrocarbon in the fermentation medium.

The said saturated dicarboxylic acid produced and accumulated in themedium can be recovered and isolated. For instance, extraction with anorganic solvent or precipitation by adjustment of the pH value of theliquid culture medium are generally employed. The liquid culture mediumcan be, if necessary, treated by an appropriate method, such ascentrifuging or filtration, to remove the microorganism. Thethus-treated liquid culture medium is then subjected to an appropriateprocedure, such as extraction with diethyl ether, or the like, afteracidification, so as efficiently to isolate the said desired product.

The said saturated dicarboxylic acid product can be identified asfollows. The liquid culture medium or the reaction liquid is madealkaline with potassium hydroxide in order to dissolve the saturateddicarboxylic acid. The solution is taken and acidified with concentratedhydrochloric acid and extracted with diethyl ether. The ether extract isthen treated with diazomethane for methylation, which is then subjectedto analysis by gas chromatography and GC-MS (gas chromatography and massspectrum measurement).

As described hereinbefore, the invention thus provides a process for thepreparation of said saturated dicarboxylic acids that at present can beprepared only with difficulty by synthetic methods, wherein amicrobiological process is applied to an said saturated hydrocarbon.

The salient feature of this invention resides in that, four yeastsbelonging to Candida vini, Candida entamophila, Candida blankii andPichia farinose which are capable of assimilating straight-chainhydrocarbons in a liquid medium containing a straight-chain hydrocarbonas a substrate to obtain a fermentation broth containing a long-chaindicarboxylic acid corresponding to said hydrocarbon.

All relevant microbial strains are described in Table 1.

TABLE 1 List of microbial strains discovered for there ability toproduce long-chain dicarboxylic acid in a liquid medium containing astraight-chain saturated hydrocarbon (tridecane) as substrate. STRAINGENOTYPE SOURCE MTCC246 Wild-type Microbial Type Culture Collection,IMTECH, Sector 39-A, Chandigarh. 160036, INDIA MTCC1387 Wild-typeMicrobial Type Culture Collection, IMTECH, Sector 39-A, Chandigarh.160036, INDIA MTCC1030 Wild-type Microbial Type Culture Collection,IMTECH, Sector 39-A, Chandigarh. 160036, INDIA MTCC624 Wild-typeMicrobial Type Culture Collection, IMTECH, Sector 39-A, Chandigarh.160036, INDIAMaterials and Methods

Microorganisms: n-alkane utilizing as well as dicarboxylic acidproducing organisms were screened from the culture stocks. 4 out of 75species screened were positive for production of said dicarboxylic acidfrom n-alkanes. Pichia farinose, Candida vini, candida entamophila,candida blankii were screened for production of brassylic acid fromtridecane.

MEDIA: The following media are employed.

Medium A: Growth Medium for Microorganisms.

3.0 g yeast extract, 3.0 g malt extract, 5.0 g Peptone, 10 g Glucose and1 L Distilled water, pH 7.0.

Medium B: Testing for Medium Production of Dicarboxylic Acid.

10 g (NH₄)₂HPO₄, 2 g K₂HPO₄, 0.3 g MgSO₄, 10 mg FeSO₄.7H₂O, 8 mgZnSO₄.7H₂0, 8 mg MnSO₄, 9 ml Tridecane and 900 ml Distilled water, pH7.0.

Medium D: Preparation of Resting Cells and Seed Culture for FermentationExperiments.

20 g D-Sorbitol, 10 g (NH₄)₂HPO₄, 2 g K₂HPO₄, 0.3 g MgSO₄, 10 mgFeSO₄.7H₂O, 8 mg ZnSO₄.7H₂0, 8 mg MnSO₄, 2 g Yeast extract, 100 μg ofBiotin and 900 ml Distilled water, pH 7.0.

Medium E: Fermentation Medium for Production of Dicarboxylic Acid.

Tridecane 5% v/v, Sodium acetate 5 g, K₂HPO₄ 2 g, MgSO₄ 0.3 g,FeSO₄.7H₂O 10 mg, ZnSO₄.7H₂0, 8 mg, MnSO₄ 8 mg, Yeast extract 2 g andDistilled water 900 ml, pH 7.0.

Cultivation

Medium A is for growing microorganisms. The said microorganisms weretransferred to medium A from slant and incubated at 30° C. for 2 days at200 rpm on shaker. 10 ml of the inoculum is transferred from medium A to100 ml of medium B in 500 ml flask, the cultivation was carried out at30° C. for 2 days at 200 rpm on shaker, to check the production ofdicarboxylic acid. For preparation of resting cells, 10 ml of inoculumwas transferred from medium B to 200 ml of medium D in 500 ml conicalflask, cultivation is carried at 30° C. for 2 days at 200 rpm. Afterculturing on a rotary shaker at 200 rpm for 2 days, the medium D withthe said organism is centrifuged at 8,000 rpm for 10 min, and the cellpellet is collected. The cell pellet of the said yeasts obtained istransferred to medium-E, fermentation medium for the production of saiddicarboxylic acid from liquid medium with said saturated hydrocarbon assubstrate.

The microorganisms usable in this invention are those belonging toCandida vini, Candida entamophila, Candida blankii and Pichia farinosehaving an ability to produce long-chain dicarboxylic acids fromstraight-chain hydrocarbons. Another object of this invention is toproduce increased yields of the said dicarboxylic acid from thestraight-chain hydrocarbons using the said microorganisms with inducers.Inducers such as H₂O₂, alkanes, combination of H₂0₂ and alkanes are usedfor increasing the yields for the production the said dicarboxylic acidfrom liquid medium with saturated hydrocarbon as substrate.

Long-chain dicarboxylic acid producing yeasts belonging to Candida vini,Candida entamophila, Candida blankii and Pichia farinose are inoculatedinto and cultured in a medium containing as substrate a straight-chainsaturated hydrocarbon, particularly one having a carbon number of 13, byadjusting the pH of the medium within the range of 6.0 to 7.0 in theinitial stage of culture and then at of 7.0 in the remaining course ofculture. The pH is preferably adjusted by addition of a neutralizingagent, such as ammonia, sodium hydroxide or potassium hydroxide, therebyproducing in a high yield a long-chain dicarboxylic acid correspondingto the hydrocarbon used as substrate of the medium. As means foraddition of a hydrocarbon (as substrate) and alkaline material, they maybe added simultaneously by previously mixing and forming them into anemulsion by a suitable method or they may be added separately from eachother. The cultivation is, moreover, carried out under aerobicconditions such as with shaking or stirring under aeration. Theseprocedures can bring about satisfactory contact between the saturatedhydrocarbon used as the starting compound, the liquid culture medium andthe air phase.

When D-biotin is introduced as a nutrient, and the said saturatedhydrocarbon, together with another carbon source, such as D-sorbitol,the microorganism grows initially on the carbon source rather than onthe saturated hydrocarbon. The resting cells medium is with D-biotin,and D-sorbitol, thus-grown microorganism then begins oxidation of thesaturated hydrocarbon in the fermentation medium.

By performing culture of said dicarboxylic acid producing yeasts in saidmedium by adjusting the pH of the medium to 6.0 to 7.0 in the initialstage of culture, it is possible to prevent impairment of thedicarboxylic acid producing ability of said yeasts which may be causedby mixing and growth of other contaminated microorganisms in the mediumin the course of culture. The period in which the culture of saiddicarboxylic acid producing yeasts is to be performed by maintaining themedium pH at from start of culture.

In the cultivation described above, the microorganism is grown in orderto oxidize the said saturated hydrocarbon so that the said saturateddicarboxylic acid accumulates in the culture medium.

The liquid culture medium is extracted with hexane to remove the mattersexisting in admixture with the long-chain dicarboxylic acid in thefermentation broth such as unreacted hydrocarbon, by-products such asmonocarboxylic acid, are effectively removed leaving the saiddicarboxylic acid in the said broth and then an alkaline solution suchas sodium hydroxide or potassium hydroxide is added to the fermentationbroth obtained by said culture to adjust pH of the solution to 10 to 13,preferably 11 to 12, to dissolve the dicarboxylic acid in saidfermentation broth. The said fermentation broth, is adjusted to pH 2.0,Suitable examples of strong mineral acids include, but are not limitedto, hydrochloric acid, sulfuric acid, phosphoric acid, and bromic acid,to precipitate the dissolved dicarboxylic acid and extracted with etherin which the long-chain dicarboxylic acid substantially exists in thedissolved state.

The obtained product was determined by gas chromatography.

GC assay procedure Retention time (min) Tri- Do- Tri- Methyl dec- dec-dec- Bras- Column ane anol anol sylate Packed Apizon-L Col. Temp. = 5.69.2 14.0 27.1 column 190° C. Final temp. = 250° C. 12.5° C./min ramping.Capillary SE 52 Column 1.5 1.7 2.0 4.1 column temp = 200° C. GCconditions were standardized on packed column and capillary columnThe Scheme for Alkane oxidation pathway is given below.

The present invention is now further described more in detail byreference to the following illustrative examples.

EXAMPLE 1 Production of Brassylic Acid by Fermentation of Tridecane withPichia farinosa

One loopful of Pichia farinose (MTCC246), from nutrient medium slantwere transferred to medium A, and incubated at 30 .degree. C. for 2 daysat 200 rpm on a rotary shaker. 10 ml of the inoculum was transferredfrom medium A to 100 ml of medium B in 500 ml flask, the cultivation wascarried out at 30 .degree. C. for 2 days at 200 rpm on shaker, this isto check the production of dicarboxylic acid. For preparation of restingcells, 10 ml of inoculum was transferred from medium B to 200 ml ofmedium D in 500 ml conical flask, cultivation is carried at 30 .degree.C. for 2 days at 200 rpm.

One aspect of the present invention provides a process for increasingthe yields of the said dicarboxylic acid from the straight-chainhydrocarbons using the said microorganism is by using inducers. 0.1%H₂O₂ and 0.1% alkane were used as Inducers in medium D. After culturingon a rotary shaker at 200 rpm for 2 days, the medium D with the saidorganism is centrifuged at 8,000 rpm for 10 min. 10.08 gm (for 200 ml ofmedium D), of Cell pellet was obtained after centrifugation of the saidbroth with said yeast. All mediums were sterilized at 121° C. for 20minutes.

The cell pellet of the said yeast obtained is transferred to medium-E,fermentation medium with 5% (v/v) of said saturated hydrocarbon(tridecane) for the production of said dicarboxylic acid (Brassylicacid) from liquid medium.

The carbon source is preferably maintained in contact with the aqueousphase and the microorganism. The oxidation is carried out at roomtemperature. A temperature in the range of 20° C.-35° C. is preferred.The pH of the medium is within the range of 6.0 to 7.0 in the initialstage of culturing and then at 7.0 in the succeeding state of culturing,the pH is preferably adjusted by addition of a neutralizing agent, suchas ammonia, sodium hydroxide or potassium hydroxide.

The liquid culture medium is extracted with hexane to remove the mattersexisting in admixture with the long-chain dicarboxylic acid in thefermentation broth such as unreacted hydrocarbon, by-products such asmonocarboxylic acid, are effectively removed leaving the saiddicarboxylic acid in the said broth and then an alkaline solution suchas sodium hydroxide or potassium hydroxide is added to the fermentationbroth obtained by said culture to adjust pH of the solution to 10 to 13,preferably 11 to 12, to dissolve the dicarboxylic acid in saidfermentation broth. The said fermentation broth, is adjusted to pH 2.0,Suitable examples of strong mineral acids include, but are not limitedto, hydrochloric acid, sulfuric acid, phosphoric acid, and bromic acid,to precipitate the dissolved dicarboxylic acid and extracted with etherin which the long-chain dicarboxylic acid substantially exists in thedissolved state.

The obtained product was determined by gas chromatography and wasconfirmed by GC-MS. As the GC-MS data of the product agreed with that ofthe authentic sample, the product was identified as brassylic acid.

Pichia farinose produces 3876.71 mg/L of brassylic acid by oxidation oftridecane with 73.81% Selectivity and the percentage of conversion is21.59.

EXAMPLE 2 Production of Brassylic Acid by Fermentation of Tridecane withCandida vini

One loopful of Candida vini (MTCC1387), from nutrient medium slant weretransferred to medium A. Cultivation was carried out by incubating at30° C. for 2 days at 200 rpm on a rotary shaker. Cultivation is carriedin the same procedure as described in Example 1.

According to the present invention, increasing the yields of the saiddicarboxylic acid from the straight-chain hydrocarbons using the saidmicroorganism is by using 0.1% alkane as Inducers in medium D. Afterculturing on a rotary shaker at 200 rpm for 2 days, the medium D withthe said organism is centrifuged at 8,000 rpm for 10 min. 3.098 gm (for200 ml of medium-D), of Cell pellet was obtained after centrifugation ofthe said broth with said yeast. All mediums were sterilized at 121° C.for 20 minutes.

The cell pellet of the said yeast obtained is transferred to medium-E,fermentation medium with 5% (v/v) of said saturated hydrocarbon for theproduction of said dicarboxylic acid from liquid medium.

Extraction of the liquid culture broth for isolation of saiddicarboxylic acid is carried in the same procedure as described inExample 1.

The obtained product was determined by gas chromatography and wasconfirmed by GC-MS. As the GC-MS data of the product agreed with that ofthe authentic sample, the product was identified as brassylic acid.

Candida vini, Produces 351.86 mg/L of brassylic acid by oxidation oftridecane with 99.18% Selectivity and the percentage of conversion is2.80.

EXAMPLE 3 Production of Brassylic Acid by Fermentation of Tridecane withCandida entamophila

One loopful of Candida entamophila (MTCC1030), from nutrient mediumslant were transferred to medium A. Cultivation was carried out at 30°C. for 2 days at 200 rpm on a rotary shaker. Cultivation is carried inthe same procedure as described in Example 1.

According to the present invention, increasing the yields of the saiddicarboxylic acid from the straight-chain hydrocarbons using the saidmicroorganism is by using 0.1% H₂O₂ and 0.1% Alkane as Inducers inmedium D. After culturing on a rotary shaker at 200 rpm for 2 days, themedium D with the said organism is centrifuged at 8,000 rpm for 10 min.6.79 gm (for 200 ml of medium-D) of Cell pellet was obtained aftercentrifugation of the said broth with said yeast. All mediums weresterilized at 121° C. for 20 minutes.

The cell pellet of the said yeast obtained is transferred to medium-E,fermentation medium with 5% (v/v) of said saturated hydrocarbon for theproduction of said Dicarboxylic acid from liquid medium.

Extraction of the liquid culture broth for isolation of saiddicarboxylic acid is carried in the same procedure as described inExample 1. The obtained product was determined by gas chromatography andwas confirmed by GC-MS. As the GC-MS data of the product agreed withthat of the authentic sample, the product was identified as brassylicacid.

Candida entamophila, Produces 778.02 mg/L of brassylic acid by oxidationof tridecane with 23.58% Selectivity and the percentage of conversion is28.70.

EXAMPLE 4 Production of Brassylic Acid by Fermentation of Tridecane withCandida blankii

One loopful of Candida blankii (MTCC 624), from nutrient medium slantwere transferred to medium A. And Cultivation was carried as incubatedat 30° C. for 2 days at 200 rpm on a rotary shaker. Cultivation iscarried in the same procedure as described in Example 1.

According to the present invention, increasing the yields of the saiddicarboxylic acid from the straight-chain hydrocarbons using the saidmicroorganism is by using 0.1% H₂O₂ and 1% Alkane as Inducers in mediumD. After culturing on a rotary shaker at 200 rpm for 2 days, the mediumD with the said organism is centrifuged at 8,000 rpm for 10 min. 6.79 gm(for 200 ml of medium-D), of Cell pellet was obtained aftercentrifugation of the said broth with said yeast. All mediums weresterilized at 121° C. for 20 minutes.

The cell pellet of the said yeast obtained is transferred to medium-E,fermentation medium with 5% (v/v) of said saturated hydrocarbon for theproduction of said dicarboxylic acid from liquid medium.

Extraction of the liquid culture broth for isolation of saiddicarboxylic acid is carried in the same procedure as described inExample 1.

The obtained product was determined by gas chromatography and wasconfirmed by GC-MS. As the GC-MS data of the product agreed with that ofthe authentic sample, the product was identified as brassylic acid.

Candida blankii, produces 274.09 mg/L of brassylic acid by oxidation oftridecane with 45.68% Selectivity and the percentage of conversion is5.22.

Table showing % Conversion and Selectivity of yeast for production ofbrassylic acid from Tridecane Yield % % Con- Culture no Species mg/LInducers Selectivity version MTCC246 Pichia 3876.71 0.1% H₂O₂ 73.8121.59 farinose MTCC1387 Candida 351.86 0.1% alk 99.18 2.80 vini MTCC1030Candida 778.02 1% H₂O₂ + 23.58 28.70 entamophila 0.1% alk MTCC624Candida 274.09 0.1% H₂O₂ + 45.68 5.22 blankii 1% hexThe Main Advantages of Present Invention are:

Aliphatic long-chain α,ω-dicarboxylic acids (DC) which can be producedby the microbial oxidation of n-alkanes are widely used as raw materialsto synthesize products such as perfumes, polymers, adhesive, macrolidantibiotics and high quality lubricants In pharmaceutical applications,1,13-dicarboxylic acid is valuable for preparation of synthetic muscone,an active ingredient in traditional Chinese medicine for treatingcoronary heart disease and inflammation of joints. The natural musconeis usually extracted from the scent glands of the adult male musk deers.Using chemical routes for the synthesis of long-chain alpha, omegadicarboxylic acids result in mixtures containing shorter chain lengths.As a result, extensive purification steps are necessary hence growinginterest shown lately for the method of producing such long-chaindicarboxylic acids according to a fermentation process by utilizing themicroorganisms. The salient feature of this invention resides in that,four yeasts belonging to Candida vini, Candida entamophila, Candidablankii and Pichia farinosa are screened capable of assimilatingstraight-chain hydrocarbons in a liquid medium containing astraight-chain hydrocarbon (tridecane) as a substrate to obtain afermentation broth containing a long-chain dicarboxylic acidcorresponding to said hydrocarbon.

Another advantage of the present invention is to produce increasedyields of the said dicarboxylic acid from the straight-chainhydrocarbons using the said microorganisms with Inducers. Inducers suchas H₂O₂, Alkanes, combination of H₂0₂ and Alkanes, Alkane are used forincreasing the yields for the production of the said dicarboxylic acidfrom liquid medium with saturated hydrocarbon as substrate.

1. A process for the preparation of one or more saturated dicarboxylicacids from one or more corresponding saturated hydrocarbons with thesame number of carbon atoms and wherein the stereostructure of thesaturated hydrocarbons is maintained, comprising culturing amicroorganism strain selected from the group consisting of Candida vini,Candida entamophila, Candida blankii and Pichia farinosa in a liquidfermentation medium containing the one or more saturated hydrocarbons assubstrate wherein one or more yield inducers selected from the groupconsisting of H₂O₂, alkanes, and mixtures thereof are added to theliquid fermentation medium in an amount effective for increasing theyield of saturated dicarboxylic acids.
 2. The process according to claim1, wherein a concentration of the saturated hydrocarbon substrate usedis between 5-15% (v/v).
 3. The process according to claim 1, wherein theliquid fermentation medium contains metal salts and organic cofactors.4. The process according to claim 1, wherein the saturated hydrocarbonused is a straight chain hydrocarbon with 13 carbon atoms and with amethyl group at both the terminals.
 5. The process according to claim 4,in which said saturated hydrocarbon used is tridecane and thecorresponding saturated dicarboxylic acid produced is Brassylic acid. 6.The process according to claim 1, in which said saturated hydrocarbonused is tridecane and the corresponding saturated dicarboxylic acidproduced is Brassylic acid wherein the culturing is effected underaerobic conditions.
 7. The process according to claim 1 in which saidsaturated hydrocarbon used is tridecane and the corresponding saturateddicarboxylic acid produced is Brassylic acid wherein the microorganismstrain is Pichia farinosa and selectivity with Pichia farinosa for thebrassylic acid by oxidation of the tridecane is 73.81% and percentage ofconversion is 21.59.
 8. The process according to claim 1 in which saidsaturated hydrocarbon used is tridecane and the corresponding saturateddicarboxylic acid produced is Brassylic acid wherein the microorganismstrain is Candida blankii and selectivity with Candida blankii for thebrassylic acid by oxidation of the tridecane is 45.68% and percentage ofconversion is 5.22.
 9. The process according to claim 1 in which saidsaturated hydrocarbon used is tridecane and the corresponding saturateddicarboxylic acid produced is Brassylic acid wherein the microorganismstrain is Candida vini and selectivity with Candida vini, for brassylicacid by oxidation of tridecane is 99.18% and percentage of conversion is2.80.
 10. The process according to claim 1 in which said saturatedhydrocarbon used is tridecane and the corresponding saturateddicarboxylic acid produced is Brassylic acid wherein the Candidaentamopila selectivity for brassylic acid by oxidation of the tridecaneis 23.58% and percentage of conversion is 28.70.
 11. The processaccording to claim 1 further comprising the steps of: (i) culturing themicroorganism strain in a growth medium for microorganisms in thepresence of a carbon source selected from n-decane and n-dodecane; (ii)transferring inoculum from the growth medium for microorganisms in step(i) to a testing medium for production of dicarboxylic acid andculturing; (iii) transferring inoculum cultivated in the testing mediumof step (ii) to a medium for preparation of resting cells and seedculture in the presence of the one or more inducers to cultivate restingcells; (iv) centrifuging the medium for preparation of resting cells andseed culture of step (iii) to obtain cell pellet of the microorganismstrain; (v) transferring the cell pellet obtained in step (iv) to afermentation medium for production of dicarboxylic acid and culturing inpresence of the saturated hydrocarbon substrate.
 12. The processaccording to claim 11, wherein in step (i) the growth medium formicroorganisms comprises 3.0 g yeast extract, 3.0 g malt extract, 5.0 gPeptone, 10 g Glucose and 1 L Distilled water.
 13. The process accordingto claim 11, wherein in step (ii) the testing medium for production ofdicarboxylic acid comprises 10 g (NH₄)₂HPO₄, 2 g K₂HPO₄, 0.3 g MgSO₄, 10mg FeSO₄.7H₂O, 8 mg ZnSO₄.7H₂O, 8 mg MnSO₄, 9 ml Tridecane and 900 mlDistilled water.
 14. The process according to claim 11, wherein in step(iii) the medium for preparation of resting cells and seed culturecomprises 20 g D-Sorbitol, 10 g (NH₄)₂HPO₄, 2 g K₂HPO₄, 0.3 g MgSO₄, 10mg FeSO₄.7H₂O, 8 mg ZnSO₄.7H₂O, 8 mg MnSO₄, 2 g Yeast extract, 100 μg ofBiotin and 900 ml Distilled water.
 15. The process according to claim11, wherein in step (v) the fermentation medium for production ofdicarboxylic acid comprises Tridecane 5% v/v, Sodium acetate 5 g, K₂HPO₄2 g, MgSO₄ 0.3 g, FeSO₄.7H₂O 10 mg, ZnSO₄.7H₂O 8 mg, MnSO₄ 8 mg, Yeastextract 2 g and Distilled water 900 ml.
 16. The process according toclaim 11, wherein in step (v) the culturing comprises biochemicaloxidation at a temperature between 20 to 35° C. for a period of up to 3days and pH in the range of 6 to
 7. 17. The process according to claim11, wherein after culturing according to step (v), dicarboxylic acid isrecovered from the fermentation medium.
 18. The process according toclaim 17, wherein after culturing, the fermentation medium is extractedwith hexane to remove unreacted hydrocarbon, monocarboxylic acid andother unwanted matter, leaving dicarboxylic acid in fermentation broth,followed by addition of an alkaline solution comprising sodium hydroxideor potassium hydroxide to the fermentation broth to adjust pH of thesolution to 10 to 13 to dissolve the dicarboxylic acid in saidfermentation broth, followed by adjusting the pH of the fermentationbroth to pH 2, using a mineral acid selected from the group consistingof hydrochloric acid, sulfuric acid, phosphoric acid, and bromic acid,to precipitate the dissolved dicarboxylic acid, followed by extractionof the dicarboxylic acid with ether.
 19. The process according to claim11, wherein the media in steps (ii), (iii) and (v) include one or moreadditional nutrients selected from the group consisting of a nitrogensource and an inorganic salt.
 20. The process according to claim 19,wherein the nitrogen source is selected from the group consisting ofpeptone, urea, ammonium phosphate, ammonium chloride, ammonium sulfateand ammonium nitrate.
 21. The process according to claim 19, wherein theinorganic salt is selected from the group consisting of phosphates,sulfates and hydrochlorides of sodium, potassium, magnesium, iron,nickel and zinc.
 22. The process according to claim 20, wherein theinorganic salt is selected from the group consisting of KH₂PO₄, K₂HPO₄,Na₂HPO₄.12H₂O, MgSO₄.7H₂O, FeSO₄.7H₂O, ZnSO₄.7H₂O and NaCl.
 23. Theprocess according to claim 11, wherein in step (iii) one or morenutrients selected from the group consisting of yeast extract, meatextract and D-biotin are added to the medium for preparation of theresting cells and seed culture.
 24. A process for producing a saturatedlong-chain dicarboxylic acid which comprises culturing a yeast strainselected from the group consisting of Candida vini, Candida entamophila,Candida blankii and Pichia farinosa, capable of assimilatingstraight-chain hydrocarbons in a liquid medium containing astraight-chain hydrocarbon as a substrate to obtain a fermentation brothcontaining a long-chain dicarboxylic acid corresponding to saidhydrocarbon, and recovering a saturated long chain dicarboxylic acid,wherein one or more yield inducers selected from the group consisting ofH₂O₂, alkanes, and mixtures thereof are added to the liquid medium in anamount effective for increasing the yield of saturated dicarboxylicacids.